Medium and matrix for long-term proliferation of cells

ABSTRACT

A cell culture medium and hydrogel matrix for long term storage and proliferation of cells is provided. The cell culture medium and hydrogel matrix include an effective amount of polar amino acids, the polar amino acids selected from the group consisting of arginine, lysine, histidine, glutamic acid, and aspartic acid. The cell culture medium comprises about 5 to about 150 mM of polar amino acids. The hydrogel matrix comprises about 3 to about 150 mM of polar amino acids. Arginine and glutamic acid are preferably supplemented in the cell culture medium. Arginine, lysine, and glutamic acid are preferably supplemented in the hydrogel matrix. A method of maintaining viability and functioning of a transplant is also provided. The method of maintaining viability of a transplant includes encapsulating the cells in a hydrogel matrix and injecting the encapsulated cells into the host organism. The matrix of the present invention may also be used to promote vascularization in a transplant site prior to injection of cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/113,437, filed Jul. 10, 1998, which is incorporated by reference inits entirety, which is a continuation-in-part of application Ser. No08/568,482, filed Dec. 7, 1995, now U.S. Pat. No. 5,834,005, which is acontinuation-in-part of application Ser. No. 08/300,429, filed Sep. 2,1994, now abandoned, which is a continuation-in-part of application Ser.No. 07/841,973, filed Feb. 24, 1992, now abandoned.

FIELD OF INVENTION

The present invention relates to a cell culture medium and matrixcomposition for preserving cell viability as well as gene expression andspecialized tissue function. The present invention also relates to amatrix capable of sustaining cell viability after injection of hormonesecreting cellular moieties into living tissue.

BACKGROUND OF THE INVENTION

New methods for treating insulin-dependant diabetes mellitus arepresently being sought. At the present time, diabetes patients testtheir blood sugar levels and inject insulin when necessary. Although itis possible to transplant a pancreas from one human to another, thesurvival rate for this procedure is only 40% at one year followingsurgery. Researchers have used isolated pancreatic islets intransplantation approaches in attempts to find a viable long termtreatment of diabetes.

The islets of Langerhans are clusters of differentiated cells sharing acommon precursor. Found in the pancreas of mammals, islets takentogether can be considered as a single endocrine organ. The isletsoccupy about 7% of the human pancreas which also contains the exocrineacinar tissue. The composition of cells in the islets differs dependingon the location of the islet in the pancreas. Central to each islet is acore of insulin secreting beta cells. Surrounding the beta cells aresomatostatin secreting delta cells, glucagon secreting alpha cells andpancreatic polypeptide containing f cells. Alpha cells tend to beconcentrated in the tail and the body of the pancreas whereas, the fcells are concentrated in the head. This distribution corresponds to theembryonic origin of alpha and f cells from dorsal and ventral primordiumof the pancreas.

Pancreatic beta cells are the only cells in which the insulin gene isexpressed and, therefore, are the sole source of metabolic insulin invertebrates. Insulin is necessary in maintaining glucose homeostasis andplays a role in the normal processing of proteins and fats. Insulinrelease can be inhibited by low levels of somatostatin and stimulated byglucagon. Without sufficient insulin to metabolize glucose,hyperglycemia occurs. Insulin-dependant diabetes mellitus is a directresult of nonfunctional islets, specifically beta cells.

Among the major obstacles in islet transplantation research is aninability to induce proliferation and to keep islets viable over time.Researchers have encountered many obstacles in attempting to curediabetes resulting from the loss of islet function. For transplantation,it is necessary to preserve islet viability as well as gene expressionand secretory function.

Pancreatic islets do not grow readily in primary cultures. However,these endocrine cells have been grown with difficulty as monolayers.This difficulty of long-term culture has not only hindered thelaboratory research for such islets, but it has also hindered attemptsto carry out physiological and even clinical studies with such islets.Therefore, there is needed a medium for the long-term proliferation ofislets. A medium for the long-term survival of cells is additionallyneeded for other cell types.

Additionally, current methods of transplantation must suppress immuneresponse by the host organism that may lead to rejection of thetransplanted cells and loss of islet function. Thus, there is also aneed in the art for a simple, non-invasive method of introducing hormonesecreting cellular moieties, such as insulin secreting pancreaticislets, into a hormone deficient organism without requiring generalimmunosuppressive agents.

SUMMARY OF THE INVENTION

A cell culture medium to promote the proliferation and long-termsurvival of cells is provided. The cell culture medium includes elevatedlevels of polar amino acids. The addition of polar amino acids to themedium enhances cell proliferation and maintains cell viability forsustained periods of time.

Additionally, a hydrogel matrix for the long-term proliferation of cellsis provided. The matrix includes elevated levels of polar amino acids.The matrix of the present invention may be used as a carrier for directinjection of cells into a host organism without significant loss of cellviability or function. Additionally, the matrix acts to shield the cellsfrom the immune system of the host organism.

Also provided are transplants capable of long-term functioning in ahost. In particular, insulin secreting transplants comprising isletcells and acinar cells are provided. The transplants of the presentinvention include the matrix and allows coexistence of islet and acinarcells with improved insulin pulsatility.

A cell culture medium for long term storage and proliferation of cellsis provided. The cell culture medium includes an effective amount ofpolar amino acids. Preferred polar amino acids are selected from, butnot limited to, the group consisting of arginine, lysine, histidine,glutamic acid, and aspartic acid. The effective amount of polar aminoacids is preferably about 5 to about 150 mM and most preferably about 10to about 64 mM. In one embodiment, the polar amino acids comprise about2 to about 60 mM of arginine and about 2 to about 60 mM of L-glutamicacid. The cells cultured in the medium may be selected from a groupconsisting of lung cells, liver cells, kidney cells, thymus cells,thyroid cells, heart cells, brain cells, pancreatic islet cells,pancreatic acinar cells, and mixtures thereof.

A hydrogel matrix for long term storage and proliferation of cellulartissue is also provided, the matrix comprising about 0.01 to about 40 mMof gelatin and an effective amount of polar amino acids. The effectiveamount of polar amino acids is preferably from about 3 to about 150 mMand most preferably about 10 to about 65 mM. In one embodiment, thepolar amino acids are selected from the group consisting of arginine,glutamic acid, lysine or mixtures thereof. Preferably, the hydrogelmatrix includes about 2 to about 60 mM of L-glutamic acid, about 1.5 toabout 10 mM of L-lysine and about 1 to about 40 mM of arginine.

A method of maintaining cell viability and functioning during storage isprovided wherein the cells are imbedded in the hydrogel matrix of thepresent invention. The matrix protects cells during storage, includingfrozen storage.

A method of maintaining viability and functioning of a transplant cellafter introduction into a host organism is also provided. The methodincludes the steps of embedding the cells in the hydrogel matrixdescribed above and injecting the embedded cells into the host organism.While the majority of the matrix liquifies and is absorbed by the host,polar moieties of the matrix attach to cell surface polar moieties, thusobscuring cell surface immune recognition proteins. Advantageously, thehydrogel matrix may be injected into a transplant site prior toinjection of the cells to encourage vascularization. The encapsulatedcells may be isolated from a different species than the host organism.

The matrix of the present invention may also be used to stimulatevascularization at a site in a host organism to treat conditionsbenefitted from an increased supply of blood. The method includescontacting the site with the matrix of the present invention wherein thematrix comprises an effective amount of polar amino acids.

A transplant for implanting in a host organism is also provided. Thetransplant comprises cells having outer surfaces encapsulated by amatrix comprising an effective amount of polar amino acids. Theeffective amount of polar amino acids may be about 3 to about 150 mM.These polar amino acids serve to enhance bonding of other polar moietiesand further obscure immune recognition proteins in a host subject. Thus,cells embedded in this enhanced hydrogel matrix substantially escapehost immune destruction.

A method for increasing insulin production in a transplant is alsoprovided. Insulin production may be increased in a transplant byproviding a mixture of acinar cells and islet cells and encapsulatingthat mixture in a matrix comprising an effective amount of polar aminoacids to form a transplant. The transplant is then injected into a hostorganism. Preferably the mixture of acinar cells and islet cellscomprises at least about 30% by volume acinar cells and most preferablyabout 60% by volume acinar cells.

A method of metabolically refeeding stored cells is also part of thepresent invention. Stored cells may be refed by providing a container ofstored cells at room temperature and adding cell culture medium of thepresent invention to the container. The container of stored cells isthen incubated for a period of time. Advantageously, the cell culturemedium is added in an amount equal to about 10 to about 40 μl/ml ofstored cells.

A method of protecting cells during isolation of the cells after enzymicdigestion of cell tissue is also included in the present invention. Themethod includes the steps of collecting digestate from a digestionprocess and adding cell culture medium of the present invention to thedigestate to protect cells during isolation.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings, wherein:

FIG. 1 is a table listing the amount of porcine c-peptide produced bythree separately designated animals;

FIG. 2 is a set of three graphs indicating the relationship betweeninsulin production and islet purity;

FIG. 3 is a table indicating insulin collection as a function of isletnumber, purity and age; and

FIG. 4 is a bar chart showing the relationship between the blood glucoselevels of two dogs.

DETAILED DESCRIPTION OF THE INVENTION

The invention comprises compositions and methods useful for making andusing transplants. The invention also comprises compositions and methodsof maintaining cell viability and function over long periods of time.Specifically, the invention provides a cell culture medium compositionand matrix composition that facilitates long-term storage andtransplantation of cells.

By transplant is intended cells, tissues, or other living or non-livingdevices for transplantation into a mammal. Transplants of the inventioninclude allografts, artificial organs, cellular transplantation andother applications for hormone producing or tissue producingimplantation into deficient individuals who suffer from conditions suchas diabetes, thyroid deficiency, growth hormone deficiency, congenitaladrenal hyperplasia, Parkinson's disease, and the like. Likewise, thematrix is useful for transplants involving therapeutic conditionsbenefitting from implantable delivery systems for biologically activeand gene therapy products for the treatment of central nervous systemdiseases and other chronic disorders. More specifically, the matrix asdescribed will find application in the various transplantationtherapies, including without limitation cells secreting human nervegrowth factors for preventing the loss of degenerating cholinergicneurons, satellite cells for myocardial regeneration, striatal braintissue for Huntington's disease, liver cells, bone marrow cells,dopamine-rich brain tissue and cells for Parkinson's disease,cholinergic-rich nervous system for Alzheimer's disease, adrenalchromaffin cells for delivering analgesics to the central nervoussystem, cultured epithelium for skin grafts, and cells releasing ciliaryneurotropic factor for amyotrophic lateral sclerosis, and the like.

Cell Culture Medium

In order to cultivate animal cells in vitro, conditions such as thosefound in vivo must be reproduced as closely as possible. Theseconditions are affected by numerous factors, including: temperature, pH,osmotic pressure, cell growth matrix, essential metabolites,supplemental metabolites, hormones, and specific factors for cellmetabolism such as transport factors, antibiotics, etc.

A medium for the long-term survival and proliferation of cells isprovided. In general, the terms “medium” and “media” in connection withthe present invention are solutions containing growth factors andnutrients which are used to support the growth and development of cells,particularly islet cells. By “long-term” is meant continuous growth anddevelopment of the cells being cultured, for a time period of at leastabout 12 to about 20 weeks, preferably greater than 20 weeks, and morepreferably greater than 40 weeks.

The medium of the invention is useful for the growth and proliferationof a variety of cells. Such cells may be derived from a variety oftissues such as lung, liver, kidney, thymus, thyroid, heart, brain,pancreas, and the like.

A number of amino acids are included within the medium. Of particularinterest are polar amino acids, particularly arginine and glutamic acid.By amino acid is intended all naturally occurring alpha amino acids inboth their D and L stereoisomeric forms, and their analogues andderivatives. An analog is defined as a substitution of an atom in theamino acid with a different atom that usually has similar properties. Aderivative is defined as an amino acid that has another molecule or atomattached to it. Derivatives would include, for example, acetylation ofan amino group, amination of a carboxyl group, or oxidation of thesulfur residues of two cysteine materials to form cystine.

The addition of supplemental amounts of polar amino acids is animportant feature of the cell culture medium of the present invention.While the invention is not bound by any particular mechanism, it isbelieved that the polar amino acids strengthen cellular membranes bybinding to polar groups found on the cellular membrane surface. Thisincreases the integrity of the cellular membrane and protects the cellfrom trauma in the culture medium environment. Additionally, the polaramino acids may bond to immune recognition sites on the cell surfacewhich suppresses adverse immune responses.

The concentration of polar amino acids may be raised until an effectiveamount of polar amino acids are present in the culture medium. Thepreferred polar amino acids are selected from the group consisting ofarginine, lysine, histidine, glutamic acid, and aspartic acid, althoughother chemicals containing polar amine and carbonyl groups may be used.An effective amount is the amount necessary to strengthen cellularmembranes and bond to immune recognition sites on the cell surface. Inone embodiment, the concentration of polar amino acids is raised to afinal concentration of between about 5 to about 150 mM, preferably about10 to about 65 mM, and more preferably about 15 to about 40 mM.

Advantageously, supplemental amounts of arginine and L-glutamic acid areadded to the culture medium of the present invention. Preferably, thefinal concentration of arginine is about 2 to about 60 mM, preferablyabout 5 to about 30 mM, most preferably about 5 to about 15 mM. Thefinal concentration of L-glutamic acid is about 2 to about 60 mM,preferably about 5 to about 30 mM, most preferably about 10 to about 20mM. In one embodiment, the final concentration of arginine is about 10mM and the final concentration of L-glutamic acid is about 15 mM.

The cell culture medium may also be used to protect cells during anisolation process following digestion of cellular tissue. By adding thecell culture medium to the digestate, the cells are protected fromdigestion, the mechanical trauma caused by the isolation process, andlater, after mixing with serum, attack by high affinity antibodies. Theresult is less cell fragmentation during isolation.

In addition to supplemental amounts of polar amino acids, the culturemedium of the present invention also comprises a standard culture mediumsupplemented with a buffering agent, salt solution and other additives.The preferred standard culture medium is Medium 199 1×liquid. However,other standard culture media known in the art would be suitable for usewith the present invention. Standard culture media which may be employedin accordance with the present invention are standard culture media forgrowing cells that typically provide an energy source, such as glucose,substantially all essential and nonessential amino acids and vitaminsand/or other cell growth supporting organic compounds required at lowconcentrations. When combined with a buffering agent and a saltsolution, the standard culture medium provides many of the nutrientsrequired for normal metabolic functioning of cultured cells.

The preferred salt solution is Earle's Balanced Salts. The salt solutionhelps to maintain pH and osmotic pressure and also provides a source ofenergy. The preferred buffering agent is Hepes. Other salt solutions andbuffering agents known in the art may be used without departing from thepresent invention.

Table 1 below lists the particularly preferred components along withpreferred approximate concentrations for each component of a solutioncontaining a standard culture medium, buffering agent and salt solution.The concentrations are based on use of Medium 199 liquid, Earle'sBalanced Salts and Hepes.

TABLE 1 Component Preferred Concentration (mM) Inorganic Salts CalciumChloride 1.26000 potassium chloride 5.34000 potassium phosphate 0.43900Magnesium sulfate * 7H2O 0.83074 Sodium chloride 90.48920 sodiumbicarbonate 4.14050 sodium phosphate 0.38500 ferric nitrate *9H2O0.00170 sodium acetate 0.62000 Amino Acids Cystine 0.08300 L-alanine0.11670 L-arginine HCl 0.39861 L-aspartic acid 0.23000 L-cysteine HCl*2H2O 0.00057 L-glutamic acid 0.51000 L-glutamine 0.68400 glycine0.53674 L-histidine HCl H2O 0.12935 L-hydroxyproline 0.07600L-isoleucine 0.33957 L-leucine 0.58299 L-lysine HCl 0.52630 L-methionine0.07424 L-phenylanine 0.17424 L-proline 0.34497 L-serine 0.28878L-threonine 0.08936 L-tryptophan 0.05860 L-tyrosine 2Na*2H2O 0.44372L-valine 0.09974 Vitamins ascorbic acid (vitamin C) 0.00233alpha-tocopherol phosphate 0.00003 d-biotin 0.00004 Dexpanthenol 0.00008choline chloride 0.00360 folic acid 0.00002 i-inositol 0.00028 menadione0.00006 niacin 0.00024 niacinamide 0.00034 para-aminobenzoic acid0.00036 pyridoxal HCl 0.00015 pyridoxine HCl 0.00023 riboflavin 0.00003Vitamin A acetate (retinol) 0.00059 Vitamin D (calciferol (ergo) 0.00054Other Additives D-glucose 4.15900 adenine sulfate 0.04300 adenosine5-triphosphate 0.00190 adenosine 5-phosphate 0.00073 cholesterol 0.00052deoxyribose 0.00370 glutathione 0.00016 guanine HCl 0.00160 HEPES25.00000 hypoxanthine sodium 0.00290 D-ribose 0.00330 thymine 0.00240tween 80 0.01500 uracil 0.00240 xanthine Na 0.00034

Advantageously, aminoguanidine may be added to the cell culture mediumof the present invention. Aminoguanidine is an L-arginine analogue andacts as a nitric oxide inhibitor. Nitric oxide and its metabolites areknown to cause cellular death from nuclear destruction and relatedinjuries. Other L-arginine analogues, such as N-monomethyl L-arginine,N-nitro-L-arginine or D-arginine could also be used in the presentinvention. Aminoguanidine is provided at a concentration of about 15 toabout 250 μM, preferably about 30 to 180 μM, most preferably about 80 toabout 120 μM. In one embodiment, the concentration of aminoguanidine isabout 100 μM.

The concentration of L-cysteine is also increased in the cell culturemedium of the present invention. L-cysteine acts as a scavenger ofalready formed nitric oxide and thereby prevents nitric oxide inducedcellular damage. Additionally, L-cysteine may obscure immune recognitionsites on the cultured cells by sulfhydryl bond formation to integralsurface proteins containing sulfur groups. Further, L-cysteine providessulfhydryl bonds which strengthen cell membranes. The preferred finalconcentration of L-cysteine is about 50 to about 300 μM, preferablyabout 80 to about 250 μM, most preferably about 150 to about 200 μM. Inone embodiment, the final concentration is about 180 μM.

Although it is possible to use the cell culture medium of the presentinvention as a serum-free medium, albumin or other nutrient sources maybe added. Use of albumin instead of conventional sera reduces cost andfacilitates transplantation of cells. It is recognized that any sourceof albumin may be used and generally human albumin is used in mostconventional culture media. For purposes of the present invention, thealbumin or serum used is preferably isolated from the same species asthe cells to be stored in the culture medium. For instance, forculturing of porcine pancreatic islet cells, porcine albumin or serumwould be used. Use of albumin from the same species as the culturedcells negates the problems of cross-species antibody attacks upon thecells and IgM cross-linking. The cultured cells are more robust whensame-species sera is used. Preferably, the concentration of albumin isabout 5 to about 50 μl/ml, preferably about 10 to about 30 μl/ml, mostpreferably about 15 μl/ml to about 25 μl/ml. In one embodiment, theconcentration of albumin is about 20 μl/ml.

Other additives known in the art may also be added to the culture mediumwithout departing from the present invention. For instance, antibioticsare preferably added to the medium. Any antibiotic known in the art maybe used. It is recognized that the antibiotic of choice may varydepending on the type of cells. Preferred antibiotics includeColy-mycin, Amphotericin b, Ciprofloxacin and Gentamicin Sulfate and thelike. The cell culture medium may also be supplemented with additionalL-glutamine to compensate for the degradation of that amino acid thatmay occur over time.

Table 2 below lists the particularly preferred additives andsupplemented ingredients for the culture medium of the present inventionand summarizes the final concentration ranges and preferred finalconcentrations for each ingredient.

TABLE 2 Components Concentration Range Preferred Concentration Albumin5-50 μl/ml 20 μl/ml L-Cysteine HCI 50-300 μM 180 μM Aminoguanidine15-250 μM 100 μM Coly-Mycin 5-20 μg/ml 10 μg/ml Amphotericin B 2-6 μM3.382 μM Ciprofloxacin 2-6 μg/ml 5 μg/ml Gentamicin Sulfate 2-6 μg/ml4.8 μg/ml L-Glutamine 5-15 μM 10 μM L-Glutamic Acid 2-60 mM 15 mMArginine HCI 2-60 mM 10 mM

Matrix

The present invention also provides a hydrogel matrix for storage andtransplantation of cells. The matrix is suitable for use with a varietyof cells including cells derived from tissue of the lung, liver, kidney,thymus, thyroid, heart, brain, pancreas, and the like. The matrix of thepresent invention provides numerous advantages over matrixes of theprior art. The matrix of the present invention is able to sustain cellsand complex clusters of cells such as islets. One advantage of thematrix is its ability to immobilize water at appropriate storagetemperatures and provide binding sites for cells that apparentlystimulate growth in terminal cell types, such as beta cells.

The matrix of the present invention also contains materials that providescaffolding for both cellular attachment and protection. This attributeof the matrix obviates the need for sera in maintaining long term cellcultures, such as long term cultures of islets, pancreatic acinartissue, hepatocytes, and erythrocytes. The matrix may be mixed withcells to form a transplant for injection into a host organism at atransplant site without the use of an additional protective carrierdevice. Transplant site is intended to mean the predetermined site wherethe transplant will be placed within the host organism. In this manner,the matrix allows transplantation of cells through a non-invasive andsimple procedure.

A surprising feature of the matrix of the present invention is that useof the matrix allows transplantation of pancreatic islet cells at lowerpurity levels. Conventionally, islet cells are utilized at high puritylevels to avoid substantial amounts of acinar cells in contact with theislet cells because of digestion of the islet cells by the acinar cells'digestive enzymes. This results in very costly and time consumingpurification methods, as well as disposal of most pancreatic tissuebecause of the presence of acinar tissue. The matrix of the presentinvention allows coexistence of acinar cells with islet cells in vitroand after transplantation in the host organism. The matrix allows theuse of cell mixtures containing as much as 70% by volume or more ofacinar cells. It is believed that the optimum range is about 30 to about40% by volume islet purity.

The unpurified pancreatic tissue also functions better than purifiedislet cells. Unpurified pancreatic tissue has been shown to exhibitinsulin pulsatility that more closely simulates the insulin pulsatilityseen in the normal functioning of pancreatic tissue of a non-diabeticorganism. The advantage of using unpurified cells is their ability tomimic normal pancreatic functions, such as insulin pulsatility. Theinsulin pulsatility of normally functioning pancreatic tissue ischaracterized by peak concentrations occurring every 5-10 minutes.

Another feature of the matrix is its ability to stimulate or enhancevascularization in surrounding tissue. Vascularization refers to theformation of blood vessels. Stimulation or enhancement ofvascularization is defined as increasing blood vessel formation andresulting blood circulation beyond that which would occur naturally. Dueto the vascularization effect, an effective amount of the matrix may beapplied to a transplant site prior to the transplant. An effectiveamount is an amount necessary to stimulate the flow of blood to thetransplant site. In this manner, the matrix improves vascularization atthe transplant site so that a blood supply is already available for thecells when the transplant occurs. However, matrix is routinely appliedto the transplant site at the time of the procedure withneovascularization occurring within 4 to 7 days. The vascularizationeffect of the matrix increases the likelihood of long-term cellviability in a host organism.

It is also recognized that the matrix may be used to treat conditionsbenefitted by increased vascularization. Such conditions include thosewhich would benefit from an increased supply of blood such as gangrene,wound sites, and general poor circulation problems. Additionally,formation of new blood vessels in the heart is critically important inprotecting the myocardium from the consequences of coronary obstruction.Injection of the matrix into ischemic myocardium may enhance thedevelopment of collaterals, accelerate the healing of necrotic tissueand prevent infarct expansion and cardiac dilatation.

The matrix is suitable for use in the transplantation of cells within atransplant device such as described in U.S. patent application Ser. No.08/568,694, which is herein incorporated by reference in its entirety. Atransplant device is any device designed to contain and protect cellstransplanted into a host organism for the production of hormones orother factors. Examples of other transplant devices suitable for usewith the matrix include those described in U.S. Pat. Nos. 5,686,091,5,676,943 and 5,550,050. However, as discussed above, the matrix may beused as the sole transplant vehicle without using such devices.

The matrix also finds use in storage of cells without loss of viabilityor specialized cell function. For long term storage, cells may be frozenin the matrix without significant loss of viability. This hasapplication in shipping blood cells, hepatocytes, pancreatic tissue,hemopoietic stem cells, bone marrow, Leydig cells, thyroid cells,pituitary cells, cardiac cells, renal cells, and others either alone orin combination, for clinical or research applications.

Current blood banking techniques allow erythrocytes to be stored foronly two months. The matrix of the present invention allows erythrocytesto remain morphologically intact for seven months. The matrix has alsobeen demonstrated to maintain the highly specialized function of cellsfor extended periods of time. Hepatocytes have maintained theirspecialized thiol transferase, albumin, and cytochrome p450 enzymes forup to 8 weeks in vitro when stored in the matrix. Drug metabolizingactivity has been maintained for at least two weeks during storage ofhepatocytes in matrix. Human red blood cells have been stored for over 8months and reconstituted by adding water without cellular lysis. A humanneuron cell line has been demonstrated to keep specific message for upto 4 weeks. The matrix thus appears to be able to keep a variety ofpartially or totally isolated cells alive and functional for extendedperiods of time.

An important feature of the matrix of the present invention is theincreased level of polar amino acid groups. The addition of polar aminoacids increases the number of hydrogen bonding moieties whichsubsequently increase the rigidity of the matrix. The increased hydrogenbonding attracts and immobilizes water. This immobilization of waterreduces cell membrane damage caused by temperature changes. It is alsobelieved that the polar amino acid groups contribute to molecularencapsulation of the cells therein and block the immune recognitionsites present on the cell surface. This characteristic allows cellsstored in the matrix to be directly injected into a host organismwithout recognition by the host organism's immune system that theinjected cells are foreign. This would allow cross-speciestransplantation of cells without immunosuppression. For example, porcinepancreatic islet cells could be injected into human hosts using thematrix of the present invention. Use of the matrix of the presentinvention obviates the need for additional protective measures toprevent a negative immune system response by the host organism.

The matrix may contain an effective amount of polar amino acids therein.The polar amino acids may be selected from the group consisting ofarginine, lysine, histidine, glutamic acid, and aspartic acid, or otheramino acids or other polar chemicals. An effective amount is the amountnecessary to increase the rigidity of the matrix and allow directinjection of the matrix with cells encapsulated therein into a hostorganism without immunosuppression. In one embodiment, the concentrationof polar amino acids is raised to a final concentration of between about3 to about 150 mM, preferably about 10 to about 65 mM, and morepreferably about 15 to about 40 mM.

Advantageously, the added polar amino acids comprise L-glutamic acid,L-lysine, and arginine. The final concentration of L-glutamic acid isabout 2 to about 60 mM, preferably about 5 to about 40 mM, mostpreferably about 10 to about 20 mM. In one embodiment, the concentrationof L-glutamic acid is about 15 mM. The final concentration of L-lysineis about 0.5 to about 30 mM, preferably about 1 to about 15 mM, mostpreferably about 1 to about 10 mM. In one embodiment, the concentrationof L-lysine is about 5.0 mM. The final concentration of arginine isabout 1 to about 40 mM, preferably about 1 to about 30, most preferablyabout 5 to about 15 mM. In one embodiment, the final concentration ofarginine is about 10 mM.

The matrix of the present invention is a combination of a gelatincomponent and a liquid composition. The gelatin acts as a substrate forcellular attachment. The preferred gelatin component is denaturedcollagen. Denatured collagen contains polar and non-polar amino acidsthat readily form a gel based on amine, carboxyl group, hydroxyl group,and sulfhydryl group interactions. The matrix is designed to be in afree flowing or liquid phase at host body temperature in order toprovide maximum diffusion across the membrane in vivo. The matrixremains in solid phase at the lower storage temperatures, such as 4° C.

Boiling or otherwise treating intact collagen to form denatured collagenbreaks covalent chemical bonds and increases the number of heatsensitive hydrogen bonds and dipole moment attractions. By replacing thecovalent chemical bonds with temperature sensitive bonds andattractions, the desired cells can be embedded in a solid matrixformulation at colder temperatures for sustained storage. Boiling orotherwise treating intact collagen breaks the tightly coiled helicaltropocollagen subunits and causes the subunits to open up into separatepeptide chains. These uncoiled strands provide multiple binding areasfor cells to attach.

The gelatin is present at a concentration of about 0.01 to about 40 mM,preferably about 0.05 to about 30 mM, most preferably about 1 to 5 mM.Advantageously, the gelatin concentration is approximately 1.6 mM. Theabove concentrations provide a solid phase at storage temperature and aliquid phase at transplant temperature.

The gelatin component of the matrix of the present invention is mixedwith a liquid composition. The liquid composition is preferably basedupon a standard culture medium, such as Medium 199, supplemented withadditives and additional amounts of some medium components, such assupplemental amounts of polar amino acids as described above.

An additional amount of L-cysteine may be added to the matrix of thepresent invention. L-cysteine acts as a nitric oxide scavenger andobscures immune recognition sites on the surface of the cells.L-cysteine also provides disulfide linkages which increases the matrix'sresistance to force and further protects the cells contained therein.The final concentration of L-cysteine is about 5 to about 500 μM,preferably about 10 to about 100 μM, most preferably about 15 to about25 μM. In one embodiment, the final concentration is about 20 μM.

Advantageously, aminoguanidine is also added to the matrix of thepresent invention. As indicated above, aminoguanidine is an L-arginineanalogue and acts as a nitric oxide inhibitor. Other L-arginineanalogues could also be used in the present invention. The finalconcentration of aminoguanidine is about 5 to about 500 μM, preferablyabout 10 to about 100 μM, most preferably about 15 to about 25 μM. Inone embodiment, the final concentration is about 20 μM.

In order to increase cell binding, intact collagen may be added in smallamounts to provide an additional binding network for the cells containedin the matrix. The final concentration of intact collagen is from about0 to about 5 mM, preferably 0 to about 2 mM, most preferably about 0.05to about 0.5 mM. In one embodiment, the concentration of intact collagenis about 0.11 mM.

Additionally, the matrix to the present invention may include a divalentchelator which increases the rigidity of the matrix by removinginhibition of —NH₂ to —COOH hydrogen bonding. The divalent chelator alsoprotects against microbial contamination of the matrix. A preferreddivalent chelator is EDTA. The concentration range for the chelator isabout 0 to about 10 mM, preferably 1 to about 8 mM, most preferablyabout 2 to about 6 mM. In a preferred embodiment, EDTA is present at aconcentration of about 4 mM. Conventional antibiotics can also be addedto further protect against microbial contamination.

As indicated above, the matrix of the present invention does not requirethe presence of sera in order to maintain long term cell cultures.However, albumin or other nutrient sources may be added to the matrix ofthe present invention if desired. Preferably, the albumin used is of thesame species as the cells contained within the matrix. As describedabove, use of the same species albumin promotes increased robustness inthe cells contained in the matrix. The concentration of albumin is about0 to about 2% by volume, preferably 0 to about 0.5% by volume, mostpreferably about 0 to about 0.1% by volume. In a preferred embodiment,the concentration of albumin is about 0.05% by volume.

The addition of high concentrations of polar amino acid enhancements, orother polar substrates, further improves the immobilization of watersuch that cells or cell combinations may be frozen to at least −20° C.without apparent morphologic or functional damage. The increasedconcentrations of L-glutamic acid, L-lysine, arginine, in addition toincreased concentrations of cysteine, result in increased denaturedconnective tissue immobilization of water at cold temperatures. Thus,the current invention demonstrates a long term cryopreservation abilitywithout the use of membrane solubilizing agents such as DMSO (DimethylSulfoxide) that are commonly used to cryopreserve isolated cells.

For long term storage, an effective amount of cryoprotectant may beadded that allows the matrix to be stored at lower temperatures withoutcellular damage. Preferably, the cryoprotectant is metabolically stableand capable of creating an inert cushion to prevent thermal expansionand contraction of cells. A preferred cryoprotectant is sulfateddextran. The cryoprotectant is present at a concentration of about 0 toabout 2 mM, preferably 0 to about 1 mM, most preferably about 0 to about0.1 mM. In one embodiment, the cryoprotectant is present in aconcentration of about 0.086 mM.

Table 3 below lists particularly preferred key components of the matrixof the present invention along with suitable concentrations as well aspreferred concentrations for each component.

TABLE 3 Components Concentration Range Preferred ConcentrationL-glutamic acid 2 to 60 mM 15 mM L-lysine .5 to 30 mM 5.0 mM Arginine 1to 40 10 mM Gelatin 0.01 to 40 mM 1.6 mM L-cysteine 5 to 500 μM 20 μMAminoguanidine 5 to 500 μM 20 μM Intact collagen 0 to 5 mM 0.11 mM EDTA0 to 10 mM 4 mM Albumin 0 to 2% by volume 0.05% by volume Dextran 0 to 2mM 0.086 mM

Matrix Preparation

Place 835 ml of Medium 199 into a stirred beaker. While stirring, heatthe solution to 50° C. Using a syringe, add 20 ml of albumin to thestirred solution. Pipette 63.28 μl of cysteine, 1 ml of L-glutamine and200 μl of aminoguanidine into the stirred beaker. Add the followinggamma irradiated dry raw materials: 120 grams of denatured collagen, 50grams of dextran, and 0.1 grams of intact collagen. Use a glass stirringrod to aid mixing of the dry materials into solution. Pipette 8 ml ofEDTA into the solution. Pipette 5 ml of L-glutamic acid, 5 ml ofL-lysine acetate, and 5 ml of arginine HCl into the stirred beaker. Notethat the solution will turn yellow. Use 10% NaOH to adjust the pH of thematrix solution to a final pH of 7.40±0.05.

Cells may be embedded in the matrix of the present invention using thefollowing procedure. Aspirate the supernatant from centrifuged cellpellets. Add a volume of cell culture medium and matrix to the cellpellets. Add a volume of matrix approximately equal to about 4 times thepellet volume. Add a volume of cell culture medium to the cell pelletsequaling approximately 0.05 times the matrix volume added. Store theencapsulated cells at refrigerated temperatures if not usingimmediately.

The present invention also provides a method of refeeding cells storedin the matrix of the present invention. Conventionally, cell culturescould not be maintained for a duration long enough to require refeedingof the cells. However, using the matrix of the present invention, cellviability may be maintained for longer periods of time, necessitatingperiodic refeeding of the cells. Additionally, bringing the cells toroom temperature periodically allows evaluation of cell function andviability and encourages the development of communication networksbetween cells.

Periodically, during refrigeration of the cell/matrix mixture, the cellsmay be refed or metabolically “walked” using the following procedure.First the stored cell/matrix mixture is retrieved from refrigeration.The mixture is examined for excess fluid. If excess fluid is present,the fluid is pipetted away and discarded. Cell culture medium is thenadded to the mixture. In one embodiment, 400 μl of the cell culturemedium of the present invention is pipetted into each 15 ml container ofthe cell/matrix mixture. The container is shaken to distribute the cellculture medium over the entire cell/matrix mixture. The container isthen capped and transferred to a 37° C. incubator. The containers areincubated for about two hours and then transferred back torefrigeration.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL EXAMPLE 1

The bottom of FIG. 1 lists the amount of porcine c-peptide in ng/ml perweek produced by a rabbit designated Rabbit 6. Rabbit 6 was part of astudy that utilized a bioartificial endocrine device containing porcineislets in the matrix of the present invention. The bioartificial devicewas taken out after 7 weeks, designated “Week O”. At that time thedevice was surgically removed, and it was discovered that the device hadruptured resulting in the porcine islets and matrix leaking into thesurrounding tissue of Rabbit 6. However, as indicated in FIG. 1, Rabbit6 continued to produce detectable levels of porcine c-peptide until week27. This suggests that the porcine islets in the matrix of the presentinvention produced a well vascularized, immunoprivileged, site withinthe tissue of the rabbit.

EXAMPLE 2

Also referring to FIG. 1, a rabbit designated as Rabbit 3 was injectedwith 3 ml of unpurified pancreatic tissue (7% islet tissue) and 2.6 mlof purified islet tissue (100% purity). Four weeks after the injection,the rabbit displayed a significant concentration of porcine c-peptideand achieved levels of up to 0.3 ng/ml during IV glucose tolerancetesting 6 weeks after the injection. The rabbit continued to produceporcine c-peptide for 13 weeks after the injection. This indicates thatunpurified pancreatic tissue functions effectively in vivo.

EXAMPLE 3

Referring again to FIG. 1, a non-diabetic dog was injected withunpurified islets (dog 22136). After injection, the dog produced 0.2ng/ml porcine c-peptide within 6 days of implant and demonstrated levelsof at least 0.1 ng/ml porcine c-peptide at all 15 blood draw pointsduring a 90 minute IV glucose tolerance test. Two other dogs wereinjected with partially purified islets. Those two dogs demonstrated 0.1ng/ml on one occasion each after 4 weeks.

This supports a finding that unpurified pancreatic tissue, after beingplaced in the matrix of the present invention, produces mature insulinproduct as measured by c-peptide much sooner and in much greaterquantity per islet than purified islets. This is an unexpected findingbecause previous findings indicated that islet transplantation requireduse of substantially purified islets to prevent digestion of the isletsby the acinar cells. However, using the matrix of the present invention,islets coexist with acinar cells without negative effect and appear toregain physiological function faster, both in terms of quantity producedand insulin pulsatility.

EXAMPLE 4

Referring to FIG. 2, three semipermeable devices consisting of the samenumber of porcine islets in enhanced matrix (containing polar aminoacids), but differing amounts of acinar tissue were perfused for 115minutes at 37° C. with a physiologic buffer containing 100 mg/dlglucose. At 115 minutes, a bolus of glucose was injected into thesolution to bring the total glucose concentration up to 300 mg/dl for aperiod of 40-60 minutes post-bolus.

The highly purified islet device (95% islet purity) released 11,000 uUinsulin/ml within 5 minutes of seeing the glucose bolus, but only hadone much smaller insulin peak of 5000 uU insulin/ml about 25 minuteslater. Slightly less purified material (85% islet purity) demonstratedthe same initial peak of insulin at 5 minutes post bolus of 11,000 uUinsulin/ml, but showed another peak of 8,000 uU insulin/ml 15 minutespost bolus, followed by small pulsations 25-45 minutes post bolus. Inthe device containing only 50% islets, there were 4 peaks over a 22minute period of between 11,000 and 12,000 uU insulin/ml demonstratingphysiologic insulin pulsatility. These data demonstrate the ability ofthe matrix to sustain partially purified pancreatic tissue and allowsuch tissue to function physiologically.

EXAMPLE 5

FIG. 3 illustrates the amount of insulin collected as a function ofnumber of islets, purity and age. With 38% purity, over two hours withonly 9.2 thousand islets, 3,550,000 uU insulin (or 3.65 units) wereproduced. When the number of islets were doubled to 18.4 thousandislets, the total insulin doubled to 8,400,000 uU insulin produced in 2hours. Similar results, though slightly lower, were obtained from isletsof only 31% purity. Of note is the extremely small volume of tissue inthe matrix required to produce such large amounts of insulin—only 0.2 or0.4 ml. The apparent optimal range is from about 30 to about 40% isletpurity.

The matrix thus has been demonstrated to improve the communication amongdifferent cell types that apparently results in a substantialimprovement of function. Thus the matrix not only protects the cellsfrom physical and immunologic trauma, it also facilitates cellularcommunication in vitro so that the cells can maintain their function asseen in vivo.

EXAMPLE 6

Two canine subjects were pancreatectomized within two weeks of eachother and were treated with injections of mixtures of Ultralente andRegular insulin twice daily. Both animals were fed identical amounts offood with Viokase added to replace pancreatic digestive enzymes. Bloodglucose values were determined in the morning and late afternoon, andexogenous insulin requirements were based on these values.

For four weeks prior to one of the dogs being injected with 8 cc of onevolume unpurified pancreatic tissue per four volumes matrix, the twodogs had statistically equivalent blood glucose determinations, andreceived the same dose of insulin twice daily. The blood glucose levelsof a dog that was not injected (darkly shaded line) and a dog that wasultimately injected intramuscularly on Day 0 (lightly shaded line) areshown in FIG. 4. The daily AM and PM blood glucose determinations areshown beginning one week prior to injection, and out for a total ofthree weeks (22 days).

FIG. 4 demonstrates that there was no statistical difference in the AMor PM blood glucose determinations during the week prior to one dogreceiving the porcine tissue injection. Beginning the day of injection,the injected dog had a statistically significant decrease in the PMblood glucose on the same insulin dose as the uninjected dog. There wasno statistical difference in the AM blood glucose during the first weekafter injection, probably reflecting the increased insulin resistancethat mammals experience in the morning due to the effects of counterregulatory hormones such as cortisol and growth hormone. Type Idiabetics generally require twice as much insulin in the AM to cover thesame ingestion of carbohydrates as they require pre-supper because ofthis AM “cortisol” effect.

Beginning seven days after the injection, blood glucose levels in theinjected dog clearly separated from those of the uninjected animal. Bothanimals had their insulin decreased 15% beginning week two. The injecteddog's glucose continued to normalize, while the uninjected dog's bloodglucose rose as expected. The injected animal continued to havestatistically significant decreased blood glucose compared to theuninjected animal over the three week period. At that point, weseparated the animals' insulin dose so that the uninjected dog could bebetter controlled.

EXAMPLE 7

The uninjected animal in Example 6 was injected with unpurified porcinepancreatic material embedded in the matrix of the present invention tofurther protect the cells from immune recognition. Approximately 8 cc ofthis material was injected intramuscularly into the previouslyuninjected dog.

Beginning that evening, the dog's blood glucose fell, and the totalinsulin dose was cut 33%. The dog went at least seven days with thechange in daily mean blood glucose and daily mean insulin dose shownbelow:

TABLE 4 Mean Daily Blood Mean total daily Glucose insulin dose Sevendays prior to injection 180 mg % 64 units Seven days post injection 101mg % 43 units

These data demonstrate that the injected porcine tissue has the effectof more than 20 units of exogenously administered insulin, since theaverage blood glucose has fallen nearly 40% and normalized on 20 unitsless insulin. The total daily insulin released in the average humansubject is approximately 0.25 units/kg body weight, or 20 units per dayin an 80 kg man. These data do not necessarily reflect 20 units ofinsulin production, since the pulsatile release of the pancreatic tissueprobably increases the animal's insulin sensitivity.

These data clearly show the ability of unpurified porcine pancreatictissue to function without the use of immunosuppression. Based on theabove figures, isolated cells from three pancreases could treat 30-50patients.

EXAMPLE 8

Islet beta cells in the matrix of the present invention were observedafter 7 days at 4° C. in the presence of a large acinar cell withdigestive granules present. The cells appeared to have normal cytoplasmand intact ultrastructure, compared to pancreatic cells kept in Medium199 under the same conditions. The islet cells in Medium 199 showedtheir cytoplasm washed out with the acinar cell releasing digestiveenzyme material.

EXAMPLE 9

Porcine liver was digested by dicing the organ into small slices, andplacing the material in collagenase for five minutes. The digestedhepatocytes, Kupfer cells, and epithelial cells were then placed in theabove matrix and kept for 10 days at 4° C. Trypan blue exclusion stainrevealed 90% viability at 10 days. In another experiment, geneexpression for albumin was measured in 77 day old cells and lidocainemetabolism measured in 13 day old porcine hepatocytes in matrix of thepresent invention.

EXAMPLE 10

Fresh whole blood from an adult male donor was centrifuged, and serumremoved. The cellular pellet was divided into two 1 ml aliquots, andplaced in either 4 ml Hanks Buffered Saline Solution or the abovematrix, and stored at 4° C. for seven months.

At the end of seven months, the cells stored in the Hanks Solution hadtotally lysed, with no cells seen under 100× light microscopy. Thematrix-containing cellular pellet was heated to 37° C., and diluted 1:1with Hanks Solution. Intact erythrocytes with biconcave morphology at100× light microscopy were present in the matrix-containing pellet.

EXAMPLE 11

Unpurified and purified porcine pancreatic tissue was digested fromfresh pancreata using standard collagenase digestion techniques. Theunpurified or gradient purified samples were placed in a matrixcontaining 5 mM lysine, 5 mM arginine, and 10 mM glutamic acid, inaddition to 180 μM cysteine, in a one part tissue volume to four partsmatrix volume, placed in polypropylene tubes, and stored from 1 day to 6weeks at −20° C. The previously frozen tissue was then thawed andstained with TSQ (N-6-methyl-8-quinolyl paratoluenesulfonamide), aflourescent zinc dye that indicates intracellular presence of insulin.

Inspection of the cells indicated appropriate morphology of both theislet tissue and digestive acinar cells in an unpurified preparationthat was frozen for 6 weeks.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing descriptions andassociated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

What is claimed is:
 1. A hydrogel matrix for long-term storage and proliferation of cellular tissue, consisting essentially of a mixture of: gelatin; dextran or sulfated dextxan; at least one polar amino acid; and optionally, one or more of intact collagen, an L-arginine analogue, L-cysteine, and a divalent chelator.
 2. A hydrogel matrix according to claim 1, wherein said gelatin is present at a concentration of about 0.01 to about 40 mM.
 3. A hydrogel matrix according to claim 1, wherein said at least one polar amino acid is present in an amount of about 3 to about 150 mM.
 4. A hydrogel matrix according to claim 3, wherein said at least one polar amino acid is present in an amount of about 10 to about 65 mM.
 5. A hydrogel matrix according to claim 1, wherein said at least one polar amino acid is selected from the group consisting of arginine, glutamic acid, lysine and mixtures thereof.
 6. A hydrogel matrix according to claim 5, wherein L-glutamic acid is present at a concentration of about 2 to about 60 mM, L-lysine is present at a concentration of about 0.5 to about 30 mM, and arginine is present at a concentration of about 1 to about 40 mM.
 7. A hydrogel matrix according to claim 6, wherein L-glutamic acid is present at a concentration of about 5 to about 40 mM, L-lysine is present at a concentration of about 1 to about 15 mM, and arginine is present at a concentration of about 1 to about 30 mM.
 8. A hydrogel matrix according to claim 1, wherein the L-cysteine is present at a concentration of about 5 to about 500 μM.
 9. A hydrogel matrix according to claim 8, wherein the L-cysteine is present at a concentration of about 15 to about 25 μM.
 10. A hydrogel matrix according to claim 1, wherein the L-arginine analogue is present at a concentration of about 5 to about 500 μM.
 11. A hydrogel matrix according to claim 10, wherein the L-arginine analogue is present at a concentration of about 15 to about 25 μM.
 12. A hydrogel matrix according to claim 10, wherein said L-arginine analogue is aminoguanidine.
 13. A hydrogel matrix according to claim 1, wherein said at least one polar amino acid is about 10 to about 20 mM of L-glutamic acid.
 14. A hydrogel matrix according to claim 1, wherein said at least one polar amino acid is about 5 to about 15 mM of arginine.
 15. A hydrogel matrix according to claim 1, wherein said at least one polar amino acid is about 1 to about 10 mM of L-lysine.
 16. A hydrogel matrix according to claim 1, wherein the divalent chelator is present at a concentration of about 1 to about 8 mM.
 17. A hydrogel matrix according to claim 1, wherein the intact collagen is present at a concentration of about 0.05 to about 0.5 mM.
 18. A hydrogel matrix according to claim 1, wherein said gelatin is denatured collagen.
 19. A hydrogel matrix according to claim 1, wherein the at least one polar amino acid is selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, and mixtures thereof.
 20. A hydrogel matrix according to claim 1, wherein said gelatin is denatured collagen.
 21. A hydrogel matrix according to claim 20, wherein said at least one polar amino acid is selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, and mixtures thereof.
 22. A hydrogel matrix according to claim 20, wherein said at least one polar amino acid is selected from the group consisting of arginine, glutamic acid, lysine and mixtures thereof.
 23. A hydrogel matrix for long-term storage and proliferation of cellular tissue, comprising a mixture of: gelatin; dextran or sulfated dextran; at least one polar amino acid; and about 1 to about 8 mM of a divalent chelator, the divalent chelator comprising EDTA.
 24. A hydrogel matrix for long-term storage and proliferation of cellular tissue comprising a mixture of: denatured collagen; dextran or sulfated dextran; an L-arginine analogue; and at least one polar amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, and mixtures thereof.
 25. A hydrogel matrix according to claim 24, wherein said at least one polar amino acid is present in an amount of about 5 to about 150 mM.
 26. A hydrogel matrix according to claim 24, comprising: about 2 to about 60 mM of L-glutamic acid; about 0.5 to about 30 mM of L-lysine; and about 1 to about 40 mM of arginine.
 27. A hydrogel matrix according to claim 24, wherein said L-arginine analogue comprises aminoguanidine.
 28. A hydrogel matrix according to claim 24, wherein said matrix comprises about 0.01 to about 40 mM of said denatured collagen.
 29. A hydrogel matrix according to claim 24, further comprising about 5 to about 500 μM of L-cysteine.
 30. A hydrogel matrix according to claim 29, wherein said matrix comprises about 15 to about 25 μM of said L-cysteine.
 31. A hydrogel matrix according to claim 24, wherein said matrix comprises about 5 to about 500 μM of said L-arginine analogue.
 32. A hydrogel matrix according to claim 31, wherein said matrix comprises about 15 to about 25 μM of said L-arginine analogue. 